1. Field of the Invention
The invention relates to an apparatus and a method for, in a diagnosis, a food inspection, or the like, measuring a material (for example, an enzyme) or an organism (for example, a cell or a microorganism) which decomposes a substrate in a solution and induces a pH-change of the solution.
2. Related Art
In the fields of medicine and food, it is requested to specifically and rapidly make a measurement on a trace amount of a material or an organism which can induce a pH-change of substrate solution. A material or an organism which can induce a pH-change of substrate solution can be detected by using a pH electrode. Generally, it cannot be insisted that a measurement using a pH electrode is sufficiently high in specificity and sensitivity. In order to improve this point, recently, an apparatus has been disclosed in which the specific molecular reconizability of an antibody and features of a minute pH electrode are utilized so as to perform a highly sensitive and rapid measurement (U.S. Pat. No. 5,674,696 and U.S. Pat. No. 5,066,582).
FIG. 4 shows the apparatus, and FIG. 5 shows a state in which the apparatus is used. Portions of the apparatus will be described. A measurement cell 1 has a cylindrical shape in which an opening is formed at each of the upper and lower ends. A through hole is formed in the peripheral wall in the vicinity of the lower end and functions as a substrate solution inlet 2. The interior of the measurement cell 1 is composed, in the sequence starting from the lower end, of a portion filled with fresh substrate solution 3 of a large diameter, a pH electrode body accommodating portion 5 of a small diameter, and a thin-tube type solid phase insertion portion 7 which is slightly larger in diameter than the pH electrode body accommodating portion 5. A pH electrode 30 is inserted into the whole of the interior of the measurement cell. A pH electrode body portion 4 of the pH electrode 30 is accommodated in the portion filled with fresh substrate solution 3 and the pH electrode body accommodating portion 5. A pH electrode sensing portion 6 of the pH electrode 30 is accommodated in the thin-tube type solid phase insertion portion 7. A pH electrode connector 13 is attached to the basal end of the pH electrode 30. The opening of the lower end of the measurement cell 1 is hermetically sealed by the pH electrode connector 13. A reference electrode 12 is attached to the pH electrode connector 13 so as to be accommodated in the portion filled with fresh substrate solution 3.
An annular groove is formed in the peripheral wall of the thin-tube type solid phase insertion portion 7 of the measurement cell 1. An O-ring 8 is fitted into the groove. The O-ring 8 is used for, when a thin-tube type solid phase 19 shown in FIG. 5 is inserted into the thin-tube type solid phase insertion portion 7, hermetically sealing a gap between the thin-tube type solid phase 19 and the peripheral wall of the thin-tube type solid phase insertion portion 7.
The upper end opening of the measurement cell 1 serves as a thin-tube type solid phase insertion port 9 through which the thin-tube type solid phase 19 shown in FIG. 5 is to be inserted into the thin-tube type solid phase insertion portion 7. The outside of the upper end of the measurement cell 1 is a substrate solution overflow portion 10 from which an overflowing substrate solution is recovered. The substrate solution which is recovered from the substrate solution overflow portion 10 reaches a waste solution vessel 16 via a substrate solution outlet 11.
By contrast, the supply of the substrate solution to the measurement cell 1 is performed by supplying a substrate solution contained in a substrate solution vessel 15, by a solution feed pump 14 via the substrate solution inlet 2.
An electric circuit unit 17 has a data processing function which performs calculations on the basis of electric signals from the pH electrode 30 and the reference electrode 12, to calculate a pH-change and supplies the calculation result to a display unit, and a drive controlling function which controls the driving of the solution feed pump 14.
The display unit 18 performs a display corresponding to the signal supplied from the electric circuit unit 17.
When urease secreted by Helicobacter pylori is to be detected by the thus configured apparatus, for example, a monoclonal antibody to urease secreted by Helicobacter pylori serving as a receptor is previously fixed to the inner and outer walls of the thin-tube type solid phase 19.
The operator then soaks one end portion of the thin-tube type solid phase 19 in a sample solution for a given time period and takes out it from the sample solution. The one end portion is accommodated in the thin-tube type solid phase insertion portion 7 through the thin-tube type solid phase insertion port 9 of the measurement cell 1. As a result, as shown in FIG. 5, a so-called coupling state in which the pH electrode sensing portion 6 is inserted into the thin-tube type solid phase 19 is established. Under this state, the operator instructs the electric circuit unit 17 to start the operation, so that the solution feed pump 14 is driven and the substrate solution is supplied to the interior of the thin-tube type solid phase 19 to wash the interior. After a predetermined time period elapses, the solution feed pump 14 is stopped and there occurs a pH-change in the substrate solution in the thin-tube type solid phase 19. The electric circuit unit 17 detects the change on the basis of the output voltage of the pH electrode 30, and the value of the detected change is displayed on the display unit 18.
However, it has been noted that there arise the following problems in such an apparatus when the thin tube of the thin-tube type solid phase 19 is coupled with the pH electrode sensing portion 6 of the pH electrode 30.
(1) In the apparatus, in order to improve the detection sensitivity and the detection speed, the distance between the inner wall of the thin tube and the sensing portion of the pH electrode must be as short as possible, and hence the inner diameters of the thin tube and the sensing portion of the pH electrode are required to be as thin as possible. However, it is difficult to produce such a thin tube and a pH electrode. Particularly, there is a limitation in the thinness of the sensing portion of a pH electrode. When a pH sensitive field effect transistor (hereinafter, referred to as xe2x80x9cpH-FETxe2x80x9d) is used as a pH electrode, for example, the sensing portion practically has a diameter of about 0.5 mm. In accordance with this diameter, the inner diameter of a thin tube can be made to be about 0.6 mm. When a thin tube has a diameter smaller than this value, it is impossible to achieve the above-mentioned coupling.
(2) In a method in which a thin tube is coupled with a pH electrode, when the thin tube has an error in dimension or shape, there is a fear that the thin tube is contacted with the pH electrode so that the pH electrode is damaged or contaminated.
It is an object of the invention to achieve a measurement which is excellent in detection sensitivity and detection speed, and also a measurement which can be performed without causing a thin tube and the sensing portion of a pH electrode to be contacted with each other.
The apparatus for measuring material which induce a pH-change of substrate solution comprises: a thin-tube type solid phase comprising a thin tube, a receptor specific for the material which can induce a pH-change of substrate solution or the organism which can induce a pH-change of substrate solution being immobilized at least to an interior of said thin tube, the material or the organism being a measuring object; a measuring cell which accommodates a substrate solution containing a substrate for the material which can induce a pH-change of substrate solution or the organism which can induce a pH-change of substrate solution; thin-tube type solid phase holding means for holding at least one end of said thin-tube type solid phase, in said cell; a pH electrode having a sensing portion which is disposed in the vicinity of said one end of said thin-tube type solid phase held in said cell, said pH electrode detecting a pH change of said substrate solution; and substrate solution moving means for moving said substrate solution in said thin-tube type solid phase held in said cell, toward said sensing portion.
When a material which can induce a pH-change of substrate solution or an organism which can induce a pH-change of substrate solution is to be measured by using the apparatus, the operator soaks one end of the thin-tube type solid phase in the sample solution. The thin-tube type solid phase is then held in the cell by the holding means. When the substrate solution is accommodated in the cell, a pH-change occurs in the substrate solution in the thin-tube type solid phase. Thereafter, the substrate solution in the thin-tube type solid phase is moved to the sensing portion of the pH electrode by the substrate solution moving means. The pH electrode detects the pH of the substrate solution at this timing.
According the apparatus for measuring material which induce a pH-change of substrate solution, in said cell, a volume of a space between a basal end of said sensing portion of said pH electrode and said one end of said thin-tube type solid phase excluding a volume occupied by said sensing portion is 10 microliters or less. When the portion has a volume of such a degree, the substrate solution can reach the sensing portion of the pH electrode without being substantially diluted.
According to the apparatus for measuring material which induce a pH-change of substrate solution, said receptor is an antibody to urease secreted by Helicobacter pylori, and said substrate solution is a solution containing urea. According to the apparatus, it is possible to measure the concentration of urease secreted by Helicobacter pylori.
According to the apparatus for measuring material which induce a pH-change of substrate solution, said pH electrode is a pH sensitive field effect transistor. The apparatus is small in size. A measurement using the apparatus can be performed with a high sensitivity and a high speed.
According to the apparatus for measuring material which induce a pH-change of substrate solution, said thin-tube type solid phase has a fibrous material in which the receptor specific to a material which can induce a pH-change of substrate solution or an organism which can induce a pH-change of substrate solution is immobilized to the interior of said thin tube, the material or the organism being a measuring object. In the apparatus, the specific surface area of the solid phase can be increased as compared with the case using a simple thin tube, whereby the detection limit for a material or an organism to be measured can be lowered.
According to the apparatus for measuring material which induce a pH-change of substrate solution, one end of a thin-tube type solid phase comprising a thin tube is soaked in a sample solution and then taken out from said sample solution, a receptor specific for a material which can induce a pH-change of substrate solution or an organism which can induce a pH-change of substrate solution being immobilized at least to an interior of said thin tube, the material or the organism being a measuring object; said one end of said thin-tube type solid phase is disposed in a substrate solution and in the vicinity of a sensing portion of a pH electrode detecting a pH-change of a solution, said substrate solution containing a substrate for the material which can induce a pH-change of substrate solution or the organism which can induce a pH-change of substrate solution; and said substrate solution in said thin-tube type solid phase after a reaction of induction of a pH-change is moved to said sensing portion of said pH electrode.
In the method, the pH electrode outputs a result which is identical with that measured by inserting a sensing portion into a thin-tube type solid phase.